The detection of the presence and/or the enumeration of absolute levels of one or more species of particles like cells or viruses in samples such as human and non-human body fluids is of primary importance in determining the state of health of human beings and mammals in general. Clinically important examples of such applications involve counting of CD4+ cells in HIV-positive subjects, of granulocytes and platelets in patients treated with chemotherapy, and of leukocytes in blood bags. Non-medical applications, on the other hand, include the detection of (bacterial) contaminants in environmental samples such as sewage or in food products.
The main analytical platform for performing such analyses is currently flow cytometry-based assay systems. Flow cytometry involves the delivery of a flowing stream containing a sample having target particles therein to the detection region of a flow cytometer. The particles are arranged in single file along a core stream using hydrodynamic focusing within a sheath fluid. The particles are then individually interrogated by a light beam. Typically, the target particles in the detection region are irradiated using a laser to create an illumination phenomenon by the target particles. The optics and detection electronics measure the light absorption, scattering, fluorescence, and/or spectral properties of the target particles in the sample, or alternatively, the respective properties of a fluorescent label attached to the target particles. In case of fluorescence, each target particle produces a burst of fluorescence photons as it passes through the illumination region. Furthermore, differentiation of the fluorescence from the illumination or the excitation light can be accomplished with a filter or a combination of filters. Detection of the fluorescence is achieved using a photomultiplier tube or a photodiode. Another technique relies on light scattering of photons in the illumination beam by the target particle. The target particle is identified by its light scattering as a function of the angle of scattering, which is, in turn, a function of its size and shape as well as the wavelength of the scattered photons.
Thus, the successful detection and identification of a single target particle depends upon several factors. First, the laser power must be sufficient to generate a large enough number of fluorescence (or alternatively scattering) photons during the brief passage of the target particle through the irradiation region. Detection of the particle typically occurs when the a sufficient number of photons is generated so that the fluorescent burst from the target particle is reliably differentiated from random fluctuations of background photons. Second, it is important to minimize these unwanted background photons arising from scattering or from fluorescence emitted by the carrier liquid of the sample or impurities in the liquid, as well as from the apparatus itself, such as the walls of the capillary through which the flow stream passes.
Flow cytometers and methods for their use in different applications are described, for example, in Sharpiro, R. M. (2002) Practical Flow Cytometry, 4th ed., Wiley-Liss, New York, N.Y.; as well as inter alia in WO 90/13368, WO 99/44037, and WO 01/59429.
However, conventional flow cytometry systems remain largely inaccessible for routine clinical use due to typically bulky instrumentation, which does not only make “on-site” measurements (e.g. bedside testing) difficult but also gives rise to high costs per analysis. Thus, there is a clear need for simpler, more compact and less expensive systems, preferably exhibiting comparable performance characteristics.
Accordingly, in recent years microfluidic techniques have been employed for the purposes of developing cytometers which require smaller sample and reagent volumes (see, e.g., Altendorf, E. et al. (1997) Sens. Actuat. 1, 531-534; Huh, D. et al. (2005) Physiol. Meas. 26, R73-R98; Dittrich, P. S., and Manz, A. (2005) Anal. Bioanal. Chem. 382, 1771-1782). Analytical instruments based on these efforts are smaller and more portable than conventional devices.
An example of such a miniaturized flow cytometer is described in the International Patent Application WO 02/10713. This device uses a non-precision fluid driver that is coupled to the sample fluid receiver and the reservoirs for supporting fluids, respectively, and controlled by a closed loop feedback path, thus enabling a more compact instrumental setup.
Instead of using flow cytometry it is also possible to determine the presence and/or number of particles in a given sample in an indirect manner by employing molecular markers (i.e. labels) that are specific for the particles of interest, and whose copy number in the sample correlates with that of the particles. Currently, different approaches are available to perform such analyses, for example ELISA-based assays (see, e.g., Kannangal, R. et al. (2001) Clin. Diagn. Lab. Immunol. 8, 1286-1288) as well as microscope-based methods involving the use of coated paramagnetic beads (see, e.g., Carella, A. Y. et al. (1995) Clin. Diagn. Lab. Immunol. 2, 623-625) or coated latex beads (see, e.g., Balakrishnan, P. et al (2004) J. Acquir. Immune Defic. Syndr. 36, 1006-1010). Furthermore, it is also possible to specifically capture the labeled particles on a membrane before imaging them using microscope optics (Rodriguez, W. R. et al. (2005) PLOS Medicine 2, e182, 663-672).
Another type of assay devices for counting particles is described in the International Patent Application WO 2005/008226. The device comprises a light source projecting light into a sub-area of a sample chip containing the particles to be analyzed labeled with a dye, and a shifter for shifting the position of the chip by a predetermined distance at every predetermined time interval relative to the object lens and the light source, respectively, in such a way that a certain area adjacent to the area photographed just before is shifted to the point where the light is incident. Therefore, sub-areas on the sample chip are photographed successively. The number of particles in each sub-area is counted and mathematically processed to calculate the total number of particles in the sample.
Furthermore, the U.S. Patent Application 2006/0024756 relates to a compact imaging cytometry device for the detection of magnetically labeled target particles or cells. For that purpose, all cells present in a biological sample to be analyzed are fluorescently labeled, but only the target cells are also magnetically labeled using a monoclonal antibody coupled to ferromagnetic beads. The labeled sample, in a chamber or cuvette, is placed between two wedge-shaped magnets to selectively move the magnetically labeled cells to the observation surface of the cuvette. An LED illuminates the cells and a CCD camera captures the images of the fluorescent light emitted by the target cells. Digital image analysis provides a count of the cells on the surface that can be related to the target cell concentration of the original sample.
However, all these devices and methods described above require comparably sophisticated detection techniques, which are expensive both in terms of initial cost and maintenance of the necessary analytical instrumentation as well as require highly trained personnel. This makes the conventional systems unsuitable for routine medical practices, “bedside” testing, or in remote locations.
Thus, there still remains a need for assay devices for the qualitative and/or quantitative detection of one or more particles in a sample, which overcome the above-mentioned limitations. In particular, there is a need for devices enabling the detection even of small amounts (i.e. numbers) of a given particle not only with high sensitivity but also in an easy-to-do and cost-efficient manner.
Furthermore, there is also a need for corresponding methods using such assay devices for the rapid and reliable detection of the presence and/or the accurate determination of the amount of one or more species of particles in a given sample. In particular, there is a need for methods that can be performed “on-site”, i.e. during or immediately after collecting the sample to be analyzed.
Accordingly, it is an object of the present invention to provide such assay devices as well as the corresponding methods using the same.